It is well known that the presence of reactions between antigens and their specific antibodies, or between other pairs of complementary substances which react specifically with each other, can be demonstrated by agglutination of insoluble particles to which one of the substances is bound. Such particles may be natural cells carrying specific receptors, antigens or antibodies, for example, or may comprise artificial "beads" of latex, glass or other suitable material. The beads are first sensitized by coating them, typically by adsorption or chemical bonding, with molecules of one reactive substance. When the sensitized cells or beads are contacted with a solution containing the complementary reactive substance, molecules of the two reactants become bound together. If the dissolved reactant has multiple valency the reaction may link the cells or beads together to form aggregates. Detection of such aggregates demonstrates presence of the reaction, and may indicate the types of reactants or receptors on the particles, or may provide a semi-quantitative measure of the concentration of the dissolved reactant.
Agglutination of cells by reaction with antibodies or the like is one of the oldest serologic procedures for the identification of bacteria and other microorganisms. It has also been widely used for blood typing and for cross matching. Such agglutination processes include variants such as indirect agglutination and inhibition of agglutination, in which additional steps are required to finally produce the agglutination that is to be measured. Such other steps typically include addition of reagents and washing, and usually follow the primary reaction of the original complementary reactants. Particle agglutination produced with the aid of such supplemental procedures is nevertheless considered to be "due to" that primary reaction, without which the supplemental procedures would be ineffective.
The present invention is applicable generally for assaying the extent of agglutination in connection with all known processes of the general type indicated above, as well as the numerous related processes derivable from them by obvious variations.
Cells or other particles may be coated, either naturally or artificially, with characteristic antigens or antibodies, and may then be caused to agglutinate by the appropriate complementary reagents. The literature includes many descriptions of the selection, preparation and/or sensitization of carrier particles to carry a wide variety of substances for reaction with specific complementary substances. Also well known are detailed procedures for carrying out such reactions to produce particle agglutination.
In general, carrier particles can be sensitized by coating with either an antigen or its complementary antibody, so that the process of agglutination can be used for assaying a test solution for either member.of such a pair of complementary substances. The procedure is also applicable, with suitable selection of the carrier particles, to virtually any substance which is capable of combining with a complementary substance in a specific manner generally similar to the immunoreactions of antigens and antibodies. For example, lectins are known to react specifically with particular carbohydrates. Either member of such pairs can, in general, be associated with the particles, whether natural or artificial, and will tend to cause agglutination upon reaction with the complementary member. Such agglutination can be used to assay either member of the pair of reactants.
Many naturally occurring carrier particles, such as lymphocytes, other cells and bacteria, have characteristic binding sites and may be quantitated directly by use of appropriate antibodies or lectins which are specific to the sites and cause the particles to agglutinate. For example, many types of bacteria carry antigens that are characteristic of the particular type. Such bacteria may be caused to agglutinate when contacted with an antibody specific to the carried antigen. That reaction may be used for identifying bacteria by the resulting agglutination when contacted with an antibody known to be specific to the antigen; or may be used for demonstrating presence in a patient's serum, say, of antibodies against a particular type of bacteria by agglutination produced when such bacteria are added to a sample of the serum. A similar reaction is widely used for typing a patient's red blood cells by observing the type of antibody that reacts with them to produce agglutination.
Moreover, it is not a requirement that detectable aggregation occur when the test reactant to be assayed is brought into contact with the complementary substance; rather, combination of the two complementary reactants may occur without aggregation at that stage, and further manipulation or addition of reagents may be needed to produce the agglutination by which presence of the test reactant is to be assayed. Illustrative of such indirect agglutination processes is the Coombs test. For example, an original reaction between an antigen carried on natural or artificial particles and a dissolved antibody may bind the antibody but fail to produce apppreciable agglutination. Conventional additional steps to produce aggregation then may include elimination of dissolved non-reactive material from the particles and their coupled reactants, as by centrifugation and washing or by chromatography or other methods; followed by addition of a supplementary reactant complementary to the antibody, such as an anti-antibody, specific to the first antibody, produced in a suitable animal. The particle agglutination produced with the aid of such supplemental procedures provides a measure of the extent of the original immunological reaction, which may be used to assay either the antigen on the particles or the antibody.
A further type of assay by indirect agglutination depends upon the ability of certain substances to inhibit agglutination. Measurement of the extent of such inhibition may be used to determine the concentration of the inhibiting substance in a test solution. For example, agglutination due to reaction of an analyte bound to insoluble particles and a specific antibody in solution is typically inhibited by presence of dissolved molecules of the analyte. The dissolved analyte reacts with the antibody in competition with the bound analyte, decreasing the effective antibody concentration available for agglutinating reaction with the bound analyte.
In actual practice, in using inhibition for analyzing a test solution for an analyte, improved sensitivity is obtained by first allowing the test solution to react with a limited amount of dissolved antibody before adding the particles with the bound analyte. If the amount of antibody in that reaction is adjusted to produce slight but clearly detectable reference agglutination when there is no analyte in the test solution and hence no inhibition, even a trace of analyte in the test solution tends to cause a relatively large percentage decrease in that slight reference agglutination. Hence, with suitable precautions, the test can be made quite sensitive. The method is especially useful for quantitation of analytes such as low molecular weight hormones or drugs which occur usually in low concentrations in serum or other fluids.
The general method of inhibition of agglutination can be used to assay in a test solution either of the complementary substances provided the test substance can be attached to, or is present on suitable particles. As in agglutination generally, the particles may be natural or artificial.
The sensitivity and speed of assay by agglutination may often be enhanced by adding a hydrophilic but immunologically inactive material such as polyethylene glycol which tends to bind water. That places the particles in a more hydrophobic environment, enhancing their tendency to bind to each other.
Furthermore, depending upon the particular system, the sensitivity may be increased and non-specific reactivity decreased if, instead of whole antibody, only an antibody fragment is used for coupling to beads, either a univalent or a bivalent fragment. Also, to minimize nonspecific reactivity of particle surfaces, such as those of glass or latex, it will be found advantageous to cover these surfaces, either by coating or coupling with non-reactive proteins or other suitable materials.
All of the above known procedures are illustrative of the broad field in which agglutination has been used. The field of usefulness of the methods of the present invention for detecting aggregate classes and measuring agglutination is correspondingly broad, and is not intended to be limited to the particular examples that have been mentioned.